162 results on '"Rinn JL"'
Search Results
2. Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2
- Author
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Marin-Bejar, O., Marchese, FP., Athie, A., Sanchez, Y., Gonzalez, J., Segura, V., Huang, L., Moreno, I., Navarro Ponz, Alfons, Monzó Planella, Mariano, Garcia-Foncillas, J., Rinn, JL., Guo, S., Huarte, M., Marin-Bejar, O., Marchese, FP., Athie, A., Sanchez, Y., Gonzalez, J., Segura, V., Huang, L., Moreno, I., Navarro Ponz, Alfons, Monzó Planella, Mariano, Garcia-Foncillas, J., Rinn, JL., Guo, S., and Huarte, M.
- Abstract
BACKGROUND: The p53 transcription factor is located at the core of a complex wiring of signaling pathways that are critical for the preservation of cellular homeostasis. Only recently it has become clear that p53 regulates the expression of several long intergenic noncoding RNAs (lincRNAs). However, relatively little is known about the role that lincRNAs play in this pathway. RESULTS: Here we characterize a lincRNA named Pint (p53 induced noncoding transcript). We show that Pint is a ubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, Pint promotes cell proliferation and survival by regulating the expression of genes of the TGF-β, MAPK and p53 pathways. Pint is a nuclear lincRNA that directly interacts with the Polycomb repressive complex 2 (PRC2), and is required for PRC2 targeting of specific genes for H3K27 tri-methylation and repression. Furthermore, Pint functional activity is highly dependent on PRC2 expression. We have also identified Pint human ortholog (PINT), which presents suggestive analogies with the murine lincRNA. PINT is similarly regulated by p53, and its expression significantly correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is downregulated in colon primary tumors, while its overexpression inhibits the proliferation of tumor cells, suggesting a possible role as tumor suppressor. CONCLUSIONS: Our results reveal a p53 autoregulatory negative mechanism where a lincRNA connects p53 activation with epigenetic silencing by PRC2. Additionally, we show analogies and differences between the murine and human orthologs, identifying a novel tumor suppressor candidate lincRNA.
- Published
- 2014
3. Sixty years of genome biology
- Author
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Doolittle, WF, Fraser, P, Gerstein, MB, Graveley, BR, Henikoff, S, Huttenhower, C, Oshlack, A, Ponting, CP, Rinn, JL, Schatz, MC, Ule, J, Weigel, D, Weinstock, GM, Doolittle, WF, Fraser, P, Gerstein, MB, Graveley, BR, Henikoff, S, Huttenhower, C, Oshlack, A, Ponting, CP, Rinn, JL, Schatz, MC, Ule, J, Weigel, D, and Weinstock, GM
- Abstract
Sixty years after Watson and Crick published the double helix model of DNA's structure, thirteen members of Genome Biology's Editorial Board select key advances in the field of genome biology subsequent to that discovery.
- Published
- 2013
4. DNMT1-interacting RNAs block gene-specific DNA methylation
- Author
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Di Ruscio, A, Ebralidze, Ak, Benoukraf, T, Amabile, G, Goff, La, Terragni, J, Figueroa, Me, De Figueiredo Pontes, Ll, Alberich Jorda, M, Zhang, P, Wu, M, D'Alo', Francesco, Melnick, A, Leone, G, Ebralidze, Kk, Pradhan, S, Rinn, Jl, Tenen, Dg, D'Alo', Francesco (ORCID:0000-0003-3576-8522), Di Ruscio, A, Ebralidze, Ak, Benoukraf, T, Amabile, G, Goff, La, Terragni, J, Figueroa, Me, De Figueiredo Pontes, Ll, Alberich Jorda, M, Zhang, P, Wu, M, D'Alo', Francesco, Melnick, A, Leone, G, Ebralidze, Kk, Pradhan, S, Rinn, Jl, Tenen, Dg, and D'Alo', Francesco (ORCID:0000-0003-3576-8522)
- Abstract
DNA methylation was first described almost a century ago; however, the rules governing its establishment and maintenance remain elusive. Here we present data demonstrating that active transcription regulates levels of genomic methylation. We identify a novel RNA arising from the CEBPA gene locus that is critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extend the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene-selective demethylation of therapeutic targets in human diseases.
- Published
- 2013
5. The Reality of Pervasive Transcription
- Author
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Eisen, MB, Clark, MB, Amaral, PP, Schlesinger, FJ, Dinger, ME, Taft, RJ, Rinn, JL, Ponting, CP, Stadler, PF, Morris, KV, Morillon, A, Rozowsky, JS, Gerstein, MB, Wahlestedt, C, Hayashizaki, Y, Carninci, P, Gingeras, TR, Mattick, JS, Eisen, MB, Clark, MB, Amaral, PP, Schlesinger, FJ, Dinger, ME, Taft, RJ, Rinn, JL, Ponting, CP, Stadler, PF, Morris, KV, Morillon, A, Rozowsky, JS, Gerstein, MB, Wahlestedt, C, Hayashizaki, Y, Carninci, P, Gingeras, TR, and Mattick, JS
- Abstract
Despite recent controversies, the evidence that the majority of the human genome is transcribed into RNA remains strong.
- Published
- 2011
6. Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2
- Author
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Universitat de Barcelona, Marin-Bejar, O., Marchese, FP., Athie, A., Sanchez, Y., Gonzalez, J., Segura, V., Huang, L., Moreno, I., Navarro Ponz, Alfons, Monzó Planella, Mariano, Garcia-Foncillas, J., Rinn, JL., Guo, S., Huarte, M., Universitat de Barcelona, Marin-Bejar, O., Marchese, FP., Athie, A., Sanchez, Y., Gonzalez, J., Segura, V., Huang, L., Moreno, I., Navarro Ponz, Alfons, Monzó Planella, Mariano, Garcia-Foncillas, J., Rinn, JL., Guo, S., and Huarte, M.
- Abstract
BACKGROUND: The p53 transcription factor is located at the core of a complex wiring of signaling pathways that are critical for the preservation of cellular homeostasis. Only recently it has become clear that p53 regulates the expression of several long intergenic noncoding RNAs (lincRNAs). However, relatively little is known about the role that lincRNAs play in this pathway. RESULTS: Here we characterize a lincRNA named Pint (p53 induced noncoding transcript). We show that Pint is a ubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, Pint promotes cell proliferation and survival by regulating the expression of genes of the TGF-β, MAPK and p53 pathways. Pint is a nuclear lincRNA that directly interacts with the Polycomb repressive complex 2 (PRC2), and is required for PRC2 targeting of specific genes for H3K27 tri-methylation and repression. Furthermore, Pint functional activity is highly dependent on PRC2 expression. We have also identified Pint human ortholog (PINT), which presents suggestive analogies with the murine lincRNA. PINT is similarly regulated by p53, and its expression significantly correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is downregulated in colon primary tumors, while its overexpression inhibits the proliferation of tumor cells, suggesting a possible role as tumor suppressor. CONCLUSIONS: Our results reveal a p53 autoregulatory negative mechanism where a lincRNA connects p53 activation with epigenetic silencing by PRC2. Additionally, we show analogies and differences between the murine and human orthologs, identifying a novel tumor suppressor candidate lincRNA.
7. Biophysical characterization of high-confidence, small human proteins.
- Author
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Whited AM, Jungreis I, Allen J, Cleveland CL, Mudge JM, Kellis M, Rinn JL, and Hough LE
- Subjects
- Humans, Biophysical Phenomena, Proteins chemistry, Proteins genetics, Open Reading Frames genetics
- Abstract
Significant efforts have been made to characterize the biophysical properties of proteins. Small proteins have received less attention because their annotation has historically been less reliable. However, recent improvements in sequencing, proteomics, and bioinformatics techniques have led to the high-confidence annotation of small open reading frames (smORFs) that encode for functional proteins, producing smORF-encoded proteins (SEPs). SEPs have been found to perform critical functions in several species, including humans. While significant efforts have been made to annotate SEPs, less attention has been given to the biophysical properties of these proteins. We characterized the distributions of predicted and curated biophysical properties, including sequence composition, structure, localization, function, and disease association of a conservative list of previously identified human SEPs. We found significant differences between SEPs and both larger proteins and control sets. In addition, we provide an example of how our characterization of biophysical properties can contribute to distinguishing protein-coding smORFs from noncoding ones in otherwise ambiguous cases., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
8. The temporal dynamics of lncRNA Firre-mediated epigenetic and transcriptional regulation.
- Author
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Much C, Lasda EL, Pereira IT, Vallery TK, Ramirez D, Lewandowski JP, Dowell RD, Smallegan MJ, and Rinn JL
- Subjects
- Humans, Gene Expression Regulation, Cell Line, Time Factors, RNA, Long Noncoding genetics, RNA, Long Noncoding metabolism, Epigenesis, Genetic, Transcription, Genetic
- Abstract
Numerous studies have now demonstrated that lncRNAs can influence gene expression programs leading to cell and organismal phenotypes. Typically, lncRNA perturbations and concomitant changes in gene expression are measured on the timescale of many hours to days. Thus, we currently lack a temporally grounded understanding of the primary, secondary, and tertiary relationships of lncRNA-mediated transcriptional and epigenetic regulation-a prerequisite to elucidating lncRNA mechanisms. To begin to address when and where a lncRNA regulates gene expression, we genetically engineer cell lines to temporally induce the lncRNA Firre. Using this approach, we are able to monitor lncRNA transcriptional regulatory events from 15 min to four days. We observe that upon induction, Firre RNA regulates epigenetic and transcriptional states in trans within 30 min. These early regulatory events result in much larger transcriptional changes after 12 h, well before current studies monitor lncRNA regulation. Moreover, Firre-mediated gene expression changes are epigenetically remembered for days. Overall, this study suggests that lncRNAs can rapidly regulate gene expression by establishing persistent epigenetic and transcriptional states., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
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9. Massively parallel dissection of RNA in RNA-protein interactions in vivo.
- Author
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Lee YH, Hass EP, Campodonico W, Lee YK, Lasda E, Shah JS, Rinn JL, and Hwang T
- Subjects
- Humans, Binding Sites, CCCTC-Binding Factor metabolism, CCCTC-Binding Factor genetics, Immunoprecipitation, Levivirus genetics, Levivirus metabolism, Mutation, Nucleic Acid Conformation, Protein Binding, RNA, Long Noncoding metabolism, RNA, Long Noncoding genetics, RNA, Long Noncoding chemistry, RNA, Viral metabolism, RNA, Viral chemistry, RNA, Viral genetics, Telomerase metabolism, Telomerase genetics, Models, Statistical, RNA metabolism, RNA chemistry, RNA genetics, RNA-Binding Proteins metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins chemistry
- Abstract
Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to biology. Here, we present massively parallel RNA assay combined with immunoprecipitation (MPRNA-IP) for in vivo high-throughput dissection of RNA-protein interactions and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, MPRNA-IP is able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest, including the long noncoding RNA NORAD, bacteriophage MS2 RNA, and human telomerase RNA, and we use it to interrogate the hitherto unknown sequence or structural RNA-binding preferences of the DNA-looping factor CTCF. By integrating systematic mutation analysis with crosslinking immunoprecipitation, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions in vivo., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
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10. Evaluation of the RNA-dependence of PRC2 binding to chromatin in human pluripotent stem cells.
- Author
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Long Y, Hwang T, Gooding AR, Goodrich KJ, Hanson SD, Vallery TK, Rinn JL, and Cech TR
- Abstract
Polycomb Repressive Complex 2 (PRC2), an important histone modifier and epigenetic repressor, has been known to interact with RNA for almost two decades. In our previous publication (Long, Hwang et al. 2020), we presented data supporting the functional importance of RNA interaction in maintaining PRC2 occupancy on chromatin, using comprehensive approaches including an RNA-binding mutant of PRC2 and an rChIP-seq assay. Recently, concerns have been expressed regarding whether the RNA-binding mutant has impaired histone methyltransferase activity and whether the rChIP-seq assay can potentially generate artifacts. Here we provide new data that support a number of our original findings. First, we found the RNA-binding mutant to be fully capable of maintaining H3K27me3 levels in human induced pluripotent stem cells. The mutant had reduced methyltransferase activity in vitro, but only on some substrates at early time points. Second, we found that our rChIP-seq method gave consistent data across antibodies and cell lines. Third, we further optimized rChIP-seq by using lower concentrations of RNase A and incorporating a catalytically inactive mutant RNase A as a control, as well as using an alternative RNase (RNase T1). The EZH2 rChIP-seq results using the optimized protocols supported our original finding that RNA interaction contributes to the chromatin occupancy of PRC2., Competing Interests: Competing Interests T.R.C. is a scientific advisor for Storm Therapeutics, Eikon Therapeutics and SomaLogic, Inc.
- Published
- 2024
- Full Text
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11. High-throughput functional analysis of regulatory variants using a massively parallel reporter assay.
- Author
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Delfosse K, Gerhardinger C, Rinn JL, and Maass PG
- Subjects
- Plasmids genetics
- Abstract
Association studies describe genetic associations between noncoding variants and disease susceptibility; however, they do not provide functional insight into the underlying molecular mechanisms of these variants. We present a protocol to assay the regulatory potential of thousands of noncoding variants using massively parallel reporter assays. We describe steps for oligo design, generating a plasmid pool, and extracting tag-seq libraries from cells to quantify the tested sequences. For complete details on the use and execution of this protocol, please refer to Oliveros and Delfosse et al.
1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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12. Natural history of Ebola virus disease in rhesus monkeys shows viral variant emergence dynamics and tissue-specific host responses.
- Author
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Normandin E, Triana S, Raju SS, Lan TCT, Lagerborg K, Rudy M, Adams GC, DeRuff KC, Logue J, Liu D, Strebinger D, Rao A, Messer KS, Sacks M, Adams RD, Janosko K, Kotliar D, Shah R, Crozier I, Rinn JL, Melé M, Honko AN, Zhang F, Babadi M, Luban J, Bennett RS, Shalek AK, Barkas N, Lin AE, Hensley LE, Sabeti PC, and Siddle KJ
- Subjects
- Animals, Macaca mulatta, Hemorrhagic Fever, Ebola pathology, Ebolavirus genetics, Hemorrhagic Fevers, Viral
- Abstract
Ebola virus (EBOV) causes Ebola virus disease (EVD), marked by severe hemorrhagic fever; however, the mechanisms underlying the disease remain unclear. To assess the molecular basis of EVD across time, we performed RNA sequencing on 17 tissues from a natural history study of 21 rhesus monkeys, developing new methods to characterize host-pathogen dynamics. We identified alterations in host gene expression with previously unknown tissue-specific changes, including downregulation of genes related to tissue connectivity. EBOV was widely disseminated throughout the body; using a new, broadly applicable deconvolution method, we found that viral load correlated with increased monocyte presence. Patterns of viral variation between tissues differentiated primary infections from compartmentalized infections, and several variants impacted viral fitness in a EBOV/Kikwit minigenome system, suggesting that functionally significant variants can emerge during early infection. This comprehensive portrait of host-pathogen dynamics in EVD illuminates new features of pathogenesis and establishes resources to study other emerging pathogens., Competing Interests: P.C.S. is a co-founder and shareholder of Sherlock Biosciences and Delve Bio, a board member and shareholder of Danaher Corporation, and has filed IP related to genomic sequencing and diagnostic technologies. A.K.S. reports compensation for consulting and/or scientific advisory board (SAB) membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Ochre Bio, Third Rock Ventures, Hovione, Relation Therapeutics, FL82, FL86, Empress Therapeutics, IntrECate Biotherapeutics, Senda Biosciences, and Dahlia Biosciences unrelated to this work. F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, and Aera Therapeutics. F.Z. is a scientific advisor for Octant., (© 2023 The Authors.)
- Published
- 2023
- Full Text
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13. Single-cell profiling of lncRNA expression during Ebola virus infection in rhesus macaques.
- Author
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Santus L, Sopena-Rios M, García-Pérez R, Lin AE, Adams GC, Barnes KG, Siddle KJ, Wohl S, Reverter F, Rinn JL, Bennett RS, Hensley LE, Sabeti PC, and Melé M
- Subjects
- Animals, Macaca mulatta, Virus Internalization, Hemorrhagic Fever, Ebola genetics, RNA, Long Noncoding genetics, Ebolavirus genetics
- Abstract
Long non-coding RNAs (lncRNAs) are involved in numerous biological processes and are pivotal mediators of the immune response, yet little is known about their properties at the single-cell level. Here, we generate a multi-tissue bulk RNAseq dataset from Ebola virus (EBOV) infected and not-infected rhesus macaques and identified 3979 novel lncRNAs. To profile lncRNA expression dynamics in immune circulating single-cells during EBOV infection, we design a metric, Upsilon, to estimate cell-type specificity. Our analysis reveals that lncRNAs are expressed in fewer cells than protein-coding genes, but they are not expressed at lower levels nor are they more cell-type specific when expressed in the same number of cells. In addition, we observe that lncRNAs exhibit similar changes in expression patterns to those of protein-coding genes during EBOV infection, and are often co-expressed with known immune regulators. A few lncRNAs change expression specifically upon EBOV entry in the cell. This study sheds light on the differential features of lncRNAs and protein-coding genes and paves the way for future single-cell lncRNA studies., (© 2023. The Author(s).)
- Published
- 2023
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14. Long non-coding RNAs: definitions, functions, challenges and recommendations.
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Mattick JS, Amaral PP, Carninci P, Carpenter S, Chang HY, Chen LL, Chen R, Dean C, Dinger ME, Fitzgerald KA, Gingeras TR, Guttman M, Hirose T, Huarte M, Johnson R, Kanduri C, Kapranov P, Lawrence JB, Lee JT, Mendell JT, Mercer TR, Moore KJ, Nakagawa S, Rinn JL, Spector DL, Ulitsky I, Wan Y, Wilusz JE, and Wu M
- Subjects
- Cell Nucleus genetics, Chromatin genetics, Regulatory Sequences, Nucleic Acid, RNA Polymerase II genetics, RNA, Long Noncoding genetics
- Abstract
Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease., (© 2023. Springer Nature Limited.)
- Published
- 2023
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15. DNMT1 inhibition by pUG-fold quadruplex RNA.
- Author
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Jansson-Fritzberg LI, Sousa CI, Smallegan MJ, Song JJ, Gooding AR, Kasinath V, Rinn JL, and Cech TR
- Subjects
- Humans, Chromatin metabolism, DNA metabolism, DNA (Cytosine-5-)-Methyltransferases antagonists & inhibitors, DNA (Cytosine-5-)-Methyltransferases metabolism, DNA Methylation, Nucleic Acid Conformation, RNA genetics, RNA metabolism, DNA (Cytosine-5-)-Methyltransferase 1 antagonists & inhibitors, DNA (Cytosine-5-)-Methyltransferase 1 metabolism
- Abstract
Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA., (© 2023 Jansson-Fritzberg et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2023
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16. T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation.
- Author
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Landshammer A, Bolondi A, Kretzmer H, Much C, Buschow R, Rose A, Wu HJ, Mackowiak SD, Braendl B, Giesselmann P, Tornisiello R, Parsi KM, Huey J, Mielke T, Meierhofer D, Maehr R, Hnisz D, Michor F, Rinn JL, and Meissner A
- Subjects
- Pregnancy, Female, Humans, Epithelial-Mesenchymal Transition, Endoderm, Gene Expression Regulation, Developmental, SOXF Transcription Factors genetics, SOXF Transcription Factors metabolism, Cell Differentiation genetics, RNA, Long Noncoding genetics, RNA, Long Noncoding metabolism
- Abstract
Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 ( T ranscript R egulating E ndoderm and activated by so X17 ) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation., Competing Interests: AL, AB, HK, CM, RB, AR, HW, SM, BB, PG, RT, KP, JH, TM, DM, RM, DH, FM, JR, AM No competing interests declared, (© 2023, Landshammer, Bolondi et al.)
- Published
- 2023
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17. Editorial overview: The genome in space-time.
- Author
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Lawrence J and Rinn JL
- Subjects
- Genome genetics
- Published
- 2022
- Full Text
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18. Prolonged FOS activity disrupts a global myogenic transcriptional program by altering 3D chromatin architecture in primary muscle progenitor cells.
- Author
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Barutcu AR, Elizalde G, Gonzalez AE, Soni K, Rinn JL, Wagers AJ, and Almada AE
- Subjects
- Cell Differentiation physiology, Chromatin genetics, Muscle Fibers, Skeletal, Stem Cells, Muscle Development, Myoblasts
- Abstract
Background: The AP-1 transcription factor, FBJ osteosarcoma oncogene (FOS), is induced in adult muscle satellite cells (SCs) within hours following muscle damage and is required for effective stem cell activation and muscle repair. However, why FOS is rapidly downregulated before SCs enter cell cycle as progenitor cells (i.e., transiently expressed) remains unclear. Further, whether boosting FOS levels in the proliferating progeny of SCs can enhance their myogenic properties needs further evaluation., Methods: We established an inducible, FOS expression system to evaluate the impact of persistent FOS activity in muscle progenitor cells ex vivo. We performed various assays to measure cellular proliferation and differentiation, as well as uncover changes in RNA levels and three-dimensional (3D) chromatin interactions., Results: Persistent FOS activity in primary muscle progenitor cells severely antagonizes their ability to differentiate and form myotubes within the first 2 weeks in culture. RNA-seq analysis revealed that ectopic FOS activity in muscle progenitor cells suppressed a global pro-myogenic transcriptional program, while activating a stress-induced, mitogen-activated protein kinase (MAPK) transcriptional signature. Additionally, we observed various FOS-dependent, chromosomal re-organization events in A/B compartments, topologically associated domains (TADs), and genomic loops near FOS-regulated genes., Conclusions: Our results suggest that elevated FOS activity in recently activated muscle progenitor cells perturbs cellular differentiation by altering the 3D chromosome organization near critical pro-myogenic genes. This work highlights the crucial importance of tightly controlling FOS expression in the muscle lineage and suggests that in states of chronic stress or disease, persistent FOS activity in muscle precursor cells may disrupt the muscle-forming process., (© 2022. The Author(s).)
- Published
- 2022
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19. Evolutionary divergence of Firre localization and expression.
- Author
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Much C, Smallegan MJ, Hwang T, Hanson SD, Dumbović G, and Rinn JL
- Subjects
- Animals, Cell Nucleus genetics, Cell Nucleus metabolism, Cytoplasm metabolism, Mice, RNA, Long Noncoding genetics, RNA, Long Noncoding metabolism
- Abstract
Long noncoding RNAs (lncRNAs) are rapidly evolving and thus typically poorly conserved in their sequences. How these sequence differences affect the characteristics and potential functions of lncRNAs with shared synteny remains unclear. Here we show that the syntenically conserved lncRNA Firre displays distinct expression and localization patterns in human and mouse. Single molecule RNA FISH reveals that in a range of cell lines, mouse Firre ( mFirre ) is predominantly nuclear, while human FIRRE ( hFIRRE ) is distributed between the cytoplasm and nucleus. This localization pattern is maintained in human/mouse hybrid cells expressing both human and mouse Firre , implying that the localization of the lncRNA is species autonomous. We find that the majority of hFIRRE transcripts in the cytoplasm are comprised of isoforms that are enriched in RRD repeats. We furthermore determine that in various tissues, mFirre is more highly expressed than its human counterpart. Our data illustrate that the rapid evolution of syntenic lncRNAs can lead to variations in lncRNA localization and abundance, which in turn may result in disparate lncRNA functions even in closely related species., (© 2022 Much et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2022
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20. From genotype to phenotype: genetics of mammalian long non-coding RNAs in vivo.
- Author
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Andergassen D and Rinn JL
- Subjects
- Animals, Gene Editing, Genome, Human, Genotype, Humans, Mammals genetics, Mice, Phenotype, RNA, Long Noncoding genetics
- Abstract
Genome-wide sequencing has led to the discovery of thousands of long non-coding RNA (lncRNA) loci in the human genome, but evidence of functional significance has remained controversial for many lncRNAs. Genetically engineered model organisms are considered the gold standard for linking genotype to phenotype. Recent advances in CRISPR-Cas genome editing have led to a rapid increase in the use of mouse models to more readily survey lncRNAs for functional significance. Here, we review strategies to investigate the physiological relevance of lncRNA loci by highlighting studies that have used genetic mouse models to reveal key in vivo roles for lncRNAs, from fertility to brain development. We illustrate how an investigative approach, starting with whole-gene deletion followed by transcription termination and/or transgene rescue strategies, can provide definitive evidence for the in vivo function of mammalian lncRNAs., (© 2021. Springer Nature Limited.)
- Published
- 2022
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21. Mosaic cis-regulatory evolution drives transcriptional partitioning of HERVH endogenous retrovirus in the human embryo.
- Author
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Carter TA, Singh M, Dumbović G, Chobirko JD, Rinn JL, and Feschotte C
- Subjects
- Animals, Genomics, Humans, Primates genetics, Terminal Repeat Sequences genetics, Endogenous Retroviruses genetics, Pluripotent Stem Cells
- Abstract
The human endogenous retrovirus type-H (HERVH) family is expressed in the preimplantation embryo. A subset of these elements are specifically transcribed in pluripotent stem cells where they appear to exert regulatory activities promoting self-renewal and pluripotency. How HERVH elements achieve such transcriptional specificity remains poorly understood. To uncover the sequence features underlying HERVH transcriptional activity, we performed a phyloregulatory analysis of the long terminal repeats (LTR7) of the HERVH family, which harbor its promoter, using a wealth of regulatory genomics data. We found that the family includes at least eight previously unrecognized subfamilies that have been active at different timepoints in primate evolution and display distinct expression patterns during human embryonic development. Notably, nearly all HERVH elements transcribed in ESCs belong to one of the youngest subfamilies we dubbed LTR7up. LTR7 sequence evolution was driven by a mixture of mutational processes, including point mutations, duplications, and multiple recombination events between subfamilies, that led to transcription factor binding motif modules characteristic of each subfamily. Using a reporter assay, we show that one such motif, a predicted SOX2/3 binding site unique to LTR7up, is essential for robust promoter activity in induced pluripotent stem cells. Together these findings illuminate the mechanisms by which HERVH diversified its expression pattern during evolution to colonize distinct cellular niches within the human embryo., Competing Interests: TC, MS, GD, JC, JR, CF No competing interests declared, (© 2022, Carter et al.)
- Published
- 2022
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22. Noncoding RNAs: biology and applications-a Keystone Symposia report.
- Author
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Cable J, Heard E, Hirose T, Prasanth KV, Chen LL, Henninger JE, Quinodoz SA, Spector DL, Diermeier SD, Porman AM, Kumar D, Feinberg MW, Shen X, Unfried JP, Johnson R, Chen CK, Wilusz JE, Lempradl A, McGeary SE, Wahba L, Pyle AM, Hargrove AE, Simon MD, Marcia M, Przanowska RK, Chang HY, Jaffrey SR, Contreras LM, Chen Q, Shi J, Mendell JT, He L, Song E, Rinn JL, Lalwani MK, Kalem MC, Chuong EB, Maquat LE, and Liu X
- Subjects
- Animals, Drug Delivery Systems methods, Drug Delivery Systems trends, Gene Targeting methods, Humans, MicroRNAs administration & dosage, MicroRNAs genetics, RNA, Long Noncoding administration & dosage, RNA, Long Noncoding genetics, RNA, Small Interfering administration & dosage, RNA, Small Interfering genetics, RNA, Small Untranslated administration & dosage, RNA, Small Untranslated genetics, Signal Transduction genetics, Congresses as Topic trends, Epigenesis, Genetic genetics, Gene Targeting trends, RNA, Untranslated administration & dosage, RNA, Untranslated genetics, Research Report
- Abstract
The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications., (© 2021 New York Academy of Sciences.)
- Published
- 2021
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23. Diverse epigenetic mechanisms maintain parental imprints within the embryonic and extraembryonic lineages.
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Andergassen D, Smith ZD, Kretzmer H, Rinn JL, and Meissner A
- Subjects
- Animals, DNA Methylation physiology, Epigenesis, Genetic genetics, Female, Histones metabolism, Mice, Pregnancy, RNA, Long Noncoding genetics, Cell Lineage physiology, Ectoderm metabolism, Genomic Imprinting genetics, Placenta metabolism
- Abstract
Genomic imprinting and X chromosome inactivation (XCI) require epigenetic mechanisms to encode allele-specific expression, but how these specific tasks are accomplished at single loci or across chromosomal scales remains incompletely understood. Here, we systematically disrupt essential epigenetic pathways within polymorphic embryos in order to examine canonical and non-canonical genomic imprinting as well as XCI. We find that DNA methylation and Polycomb group repressors are indispensable for autosomal imprinting, albeit at distinct gene sets. Moreover, the extraembryonic ectoderm relies on a broader spectrum of imprinting mechanisms, including non-canonical targeting of maternal endogenous retrovirus (ERV)-driven promoters by the H3K9 methyltransferase G9a. We further identify Polycomb-dependent and -independent gene clusters on the imprinted X chromosome, which appear to reflect distinct domains of Xist-mediated suppression. From our data, we assemble a comprehensive inventory of the epigenetic pathways that maintain parent-specific imprinting in eutherian mammals, including an expanded view of the placental lineage., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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24. Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency.
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Haswell JR, Mattioli K, Gerhardinger C, Maass PG, Foster DJ, Peinado P, Wang X, Medina PP, Rinn JL, and Slack FJ
- Subjects
- CRISPR-Cas Systems, Forkhead Transcription Factors genetics, Humans, RNA Interference, RNA, Long Noncoding, Cell Differentiation genetics
- Abstract
Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
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25. Publisher Correction: Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA is driven by intron retention.
- Author
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Dumbović G, Braunschweig U, Langner HK, Smallegan M, Biayna J, Hass EP, Jastrzebska K, Blencowe B, Cech TR, Caruthers MH, and Rinn JL
- Published
- 2021
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26. Genome-wide binding analysis of 195 DNA binding proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation.
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Smallegan MJ, Shehata S, Spradlin SF, Swearingen A, Wheeler G, Das A, Corbet G, Nebenfuehr B, Ahrens D, Tauber D, Lennon S, Choi K, Huynh T, Wieser T, Schneider K, Bradshaw M, Basken J, Lai M, Read T, Hynes-Grace M, Timmons D, Demasi J, and Rinn JL
- Subjects
- Humans, K562 Cells, Reproducibility of Results, Computational Biology methods, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Gene Expression Regulation, Genome, Human, Promoter Regions, Genetic, Repetitive Sequences, Nucleic Acid
- Abstract
A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. Here we wanted to take a deep analysis of DNA binding events and transcriptional output of a single cell state (K562 cells). To this end we re-analyzed 195 DNA binding proteins contained in ENCODE data. We used standardized analysis pipelines, containerization, and literate programming with R Markdown for reproducibility and rigor. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODE's goals in providing these reproducible data resources. We also had several new findings including: (i) 1,362 promoters, which we refer to as 'reservoirs,' that are defined by having up to 111 different DNA binding-proteins localized on one promoter, yet do not have any expression of steady-state RNA (ii) Reservoirs do not overlap super-enhancer annotations and distinct have distinct properties from super-enhancers. (iii) The human specific SVA repeat element may have been co-opted for enhancer regulation and is highly transcribed in PRO-seq and RNA-seq. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 -Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment., Competing Interests: JB, ML, and TR are employed by Arpeggio Biosciences. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents or products associated with this research. None of the other authors have any competing interests to declare.
- Published
- 2021
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27. Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA is driven by intron retention.
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Dumbović G, Braunschweig U, Langner HK, Smallegan M, Biayna J, Hass EP, Jastrzebska K, Blencowe B, Cech TR, Caruthers MH, and Rinn JL
- Subjects
- Animals, Cell Compartmentation, Cell Line, Cell Line, Tumor, HCT116 Cells, HEK293 Cells, HeLa Cells, Human Embryonic Stem Cells cytology, Human Embryonic Stem Cells metabolism, Humans, In Situ Hybridization, Fluorescence, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Introns, Mice, Mitosis, RNA Precursors genetics, RNA Precursors metabolism, RNA Splicing, RNA Stability, Species Specificity, Cell Nucleus genetics, Cell Nucleus metabolism, RNA, Long Noncoding genetics, RNA, Long Noncoding metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Telomerase genetics
- Abstract
The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.
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- 2021
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28. An in vivo screen of noncoding loci reveals that Daedalus is a gatekeeper of an Ikaros-dependent checkpoint during haematopoiesis.
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Harman CCD, Bailis W, Zhao J, Hill L, Qu R, Jackson RP, Shyer JA, Steach HR, Kluger Y, Goff LA, Rinn JL, Williams A, Henao-Mejia J, and Flavell RA
- Subjects
- Animals, Cell Differentiation genetics, Cell Lineage genetics, DNA-Binding Proteins genetics, Gene Deletion, Gene Expression Regulation, Developmental genetics, Mice, Hematopoiesis genetics, Ikaros Transcription Factor genetics, Lymphopoiesis genetics, RNA, Untranslated genetics
- Abstract
Haematopoiesis relies on tightly controlled gene expression patterns as development proceeds through a series of progenitors. While the regulation of hematopoietic development has been well studied, the role of noncoding elements in this critical process is a developing field. In particular, the discovery of new regulators of lymphopoiesis could have important implications for our understanding of the adaptive immune system and disease. Here we elucidate how a noncoding element is capable of regulating a broadly expressed transcription factor, Ikaros, in a lymphoid lineage-specific manner, such that it imbues Ikaros with the ability to specify the lymphoid lineage over alternate fates. Deletion of the Daedalus locus, which is proximal to Ikaros, led to a severe reduction in early lymphoid progenitors, exerting control over the earliest fate decisions during lymphoid lineage commitment. Daedalus locus deletion led to alterations in Ikaros isoform expression and a significant reduction in Ikaros protein. The Daedalus locus may function through direct DNA interaction as Hi-C analysis demonstrated an interaction between the two loci. Finally, we identify an Ikaros-regulated erythroid-lymphoid checkpoint that is governed by Daedalus in a lymphoid-lineage-specific manner. Daedalus appears to act as a gatekeeper of Ikaros's broad lineage-specifying functions, selectively stabilizing Ikaros activity in the lymphoid lineage and permitting diversion to the erythroid fate in its absence. These findings represent a key illustration of how a transcription factor with broad lineage expression must work in concert with noncoding elements to orchestrate hematopoietic lineage commitment., Competing Interests: Competing interest statement: R.A.F. is a consultant for GSK and Zai Lab Ltd.
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- 2021
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29. Identification of human long noncoding RNAs associated with nonalcoholic fatty liver disease and metabolic homeostasis.
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Ruan X, Li P, Ma Y, Jiang CF, Chen Y, Shi Y, Gupta N, Seifuddin F, Pirooznia M, Ohnishi Y, Yoneda N, Nishiwaki M, Dumbovic G, Rinn JL, Higuchi Y, Kawai K, Suemizu H, and Cao H
- Subjects
- Animals, Humans, Mice, Databases, Nucleic Acid, Homeostasis genetics, Non-alcoholic Fatty Liver Disease genetics, Non-alcoholic Fatty Liver Disease metabolism, RNA, Long Noncoding genetics, RNA, Long Noncoding metabolism
- Abstract
A growing number of long noncoding RNAs (lncRNAs) have emerged as vital metabolic regulators. However, most human lncRNAs are nonconserved and highly tissue specific, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). In this study, we established a pipeline to identify putative hLMRs that are metabolically sensitive, disease relevant, and population applicable. We first progressively processed multilevel human transcriptome data to select liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a nonalcoholic fatty liver disease (NAFLD) population, and respond to dietary intervention in a small NAFLD cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and NAFLD populations, we predicted the specific function for each hLMR. Using gain- and loss-of-function approaches in humanized mice as well as ectopic expression in conventional mice, we validated the regulatory role of one nonconserved hLMR in cholesterol metabolism by coordinating with an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. Our work overcame the heterogeneity intrinsic to human data to enable the efficient identification and functional definition of disease-relevant human lncRNAs in metabolic homeostasis.
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- 2021
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30. Defective insulin receptor signaling in hPSCs skews pluripotency and negatively perturbs neural differentiation.
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Teo AKK, Nguyen L, Gupta MK, Lau HH, Loo LSW, Jackson N, Lim CS, Mallard W, Gritsenko MA, Rinn JL, Smith RD, Qian WJ, and Kulkarni RN
- Subjects
- Cell Differentiation physiology, Cell Line, Cells, Cultured, Human Embryonic Stem Cells metabolism, Humans, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Neurons metabolism, Octamer Transcription Factor-3 metabolism, Phosphorylation, Pluripotent Stem Cells metabolism, Proteomics methods, Signal Transduction, Human Embryonic Stem Cells cytology, Neurons cytology, Pluripotent Stem Cells cytology, Receptor, Insulin metabolism
- Abstract
Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays, and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Of importance, RNA sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry-based proteomic analyses further confirmed a global downregulation of extracellular matrix proteins. Subsequent differentiation toward the neural lineage reflected alterations in SOX1
+ PAX6+ neuroectoderm and FOXG1+ cortical neuron marker expression and protein localization. Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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31. Myosin 7b is a regulatory long noncoding RNA (lncMYH7b) in the human heart.
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Broadwell LJ, Smallegan MJ, Rigby KM, Navarro-Arriola JS, Montgomery RL, Rinn JL, and Leinwand LA
- Subjects
- Cardiac Myosins chemistry, Humans, Induced Pluripotent Stem Cells, MicroRNAs genetics, Molecular Dynamics Simulation, Myocardium cytology, Myocytes, Cardiac metabolism, Myosin Heavy Chains chemistry, Protein Conformation, Cardiac Myosins metabolism, Myocardium metabolism, Myosin Heavy Chains metabolism, RNA, Long Noncoding genetics
- Abstract
Myosin heavy chain 7b (MYH7b) is an ancient member of the myosin heavy chain motor protein family that is expressed in striated muscles. In mammalian cardiac muscle, MYH7b RNA is expressed along with two other myosin heavy chains, β-myosin heavy chain (β-MyHC) and α-myosin heavy chain (α-MyHC). However, unlike β-MyHC and α-MyHC, which are maintained in a careful balance at the protein level, the MYH7b locus does not produce a full-length protein in the heart due to a posttranscriptional exon-skipping mechanism that occurs in a tissue-specific manner. Whether this locus has a role in the heart beyond producing its intronic microRNA, miR-499, was unclear. Using cardiomyocytes derived from human induced pluripotent stem cells as a model system, we found that the noncoding exon-skipped RNA (lncMYH7b) affects the transcriptional landscape of human cardiomyocytes, independent of miR-499. Specifically, lncMYH7b regulates the ratio of β-MyHC to α-MyHC, which is crucial for cardiac contractility. We also found that lncMYH7b regulates beat rate and sarcomere formation in cardiomyocytes. This regulation is likely achieved through control of a member of the TEA domain transcription factor family (TEAD3, which is known to regulate β-MyHC). Therefore, we conclude that this ancient gene has been repurposed by alternative splicing to produce a regulatory long-noncoding RNA in the human heart that affects cardiac myosin composition., Competing Interests: Conflict of interest No conflicts of interest were reported., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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32. Trans- and cis-acting effects of Firre on epigenetic features of the inactive X chromosome.
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Fang H, Bonora G, Lewandowski JP, Thakur J, Filippova GN, Henikoff S, Shendure J, Duan Z, Rinn JL, Deng X, Noble WS, and Disteche CM
- Subjects
- Alleles, Animals, Base Sequence, Cell Line, Cell Nucleus genetics, Chromatin metabolism, DNA, Complementary genetics, Female, Gene Deletion, Gene Ontology, Genetic Loci, Genome, Histones metabolism, Lysine metabolism, Male, Methylation, Mice, Inbred C57BL, Polycomb Repressive Complex 2 metabolism, RNA, Long Noncoding metabolism, Transgenes, Up-Regulation genetics, X Chromosome genetics, Epigenesis, Genetic, RNA, Long Noncoding genetics, X Chromosome Inactivation genetics
- Abstract
Firre encodes a lncRNA involved in nuclear organization. Here, we show that Firre RNA expressed from the active X chromosome maintains histone H3K27me3 enrichment on the inactive X chromosome (Xi) in somatic cells. This trans-acting effect involves SUZ12, reflecting interactions between Firre RNA and components of the Polycomb repressive complexes. Without Firre RNA, H3K27me3 decreases on the Xi and the Xi-perinucleolar location is disrupted, possibly due to decreased CTCF binding on the Xi. We also observe widespread gene dysregulation, but not on the Xi. These effects are measurably rescued by ectopic expression of mouse or human Firre/FIRRE transgenes, supporting conserved trans-acting roles. We also find that the compact 3D structure of the Xi partly depends on the Firre locus and its RNA. In common lymphoid progenitors and T-cells Firre exerts a cis-acting effect on maintenance of H3K27me3 in a 26 Mb region around the locus, demonstrating cell type-specific trans- and cis-acting roles of this lncRNA.
- Published
- 2020
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33. Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics.
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Kotliar D, Lin AE, Logue J, Hughes TK, Khoury NM, Raju SS, Wadsworth MH 2nd, Chen H, Kurtz JR, Dighero-Kemp B, Bjornson ZB, Mukherjee N, Sellers BA, Tran N, Bauer MR, Adams GC, Adams R, Rinn JL, Melé M, Schaffner SF, Nolan GP, Barnes KG, Hensley LE, McIlwain DR, Shalek AK, Sabeti PC, and Bennett RS
- Subjects
- Animals, Antigens, CD metabolism, Biomarkers metabolism, Bystander Effect, Cell Differentiation, Cell Proliferation, Cytokines metabolism, Ebolavirus genetics, Endoplasmic Reticulum Chaperone BiP, Gene Expression Profiling, Gene Expression Regulation, Gene Expression Regulation, Viral, Hemorrhagic Fever, Ebola immunology, Hemorrhagic Fever, Ebola pathology, Histocompatibility Antigens Class II metabolism, Interferons genetics, Interferons metabolism, Macaca mulatta, Macrophages metabolism, Monocytes metabolism, Myelopoiesis, RNA, Messenger genetics, RNA, Messenger metabolism, Time Factors, Transcriptome genetics, Ebolavirus physiology, Hemorrhagic Fever, Ebola genetics, Hemorrhagic Fever, Ebola virology, Host-Pathogen Interactions genetics, Single-Cell Analysis
- Abstract
Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment., Competing Interests: Declaration of Interests A.K.S. has received compensation for consulting and SAB membership from Honeycomb Biotechnologies, Cellarity, Ochre Bio, Repertoire, and Dahlia Biosciences., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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34. The Tug1 lncRNA locus is essential for male fertility.
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Lewandowski JP, Dumbović G, Watson AR, Hwang T, Jacobs-Palmer E, Chang N, Much C, Turner KM, Kirby C, Rubinstein ND, Groff AF, Liapis SC, Gerhardinger C, Bester A, Pandolfi PP, Clohessy JG, Hoekstra HE, Sauvageau M, and Rinn JL
- Subjects
- Animals, Gene Expression Regulation, Humans, Male, Mice, Mice, Knockout, Open Reading Frames, Spermatogenesis genetics, Fertility genetics, RNA, Long Noncoding genetics
- Abstract
Background: Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function., Results: We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function., Conclusions: Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.
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- 2020
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35. RNA is essential for PRC2 chromatin occupancy and function in human pluripotent stem cells.
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Long Y, Hwang T, Gooding AR, Goodrich KJ, Rinn JL, and Cech TR
- Subjects
- Binding Sites genetics, Carrier Proteins, Cell Differentiation genetics, Genome genetics, Histones genetics, Humans, Protein Binding genetics, Chromatin genetics, Induced Pluripotent Stem Cells physiology, Pluripotent Stem Cells physiology, Polycomb Repressive Complex 2 genetics, RNA genetics
- Abstract
Many chromatin-binding proteins and protein complexes that regulate transcription also bind RNA. One of these, Polycomb repressive complex 2 (PRC2), deposits the H3K27me3 mark of facultative heterochromatin and is required for stem cell differentiation. PRC2 binds RNAs broadly in vivo and in vitro. Yet, the biological importance of this RNA binding remains unsettled. Here, we tackle this question in human induced pluripotent stem cells by using multiple complementary approaches. Perturbation of RNA-PRC2 interaction by RNase A, by a chemical inhibitor of transcription or by an RNA-binding-defective mutant all disrupted PRC2 chromatin occupancy and localization genome wide. The physiological relevance of PRC2-RNA interactions is further underscored by a cardiomyocyte differentiation defect upon genetic disruption. We conclude that PRC2 requires RNA binding for chromatin localization in human pluripotent stem cells and in turn for defining cellular state.
- Published
- 2020
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36. Cis and trans effects differentially contribute to the evolution of promoters and enhancers.
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Mattioli K, Oliveros W, Gerhardinger C, Andergassen D, Maass PG, Rinn JL, and Melé M
- Subjects
- Animals, Conserved Sequence, Genes, Reporter, Humans, Mice, Regulatory Elements, Transcriptional, Transcription Factors, Enhancer Elements, Genetic, Evolution, Molecular, Gene Expression Regulation, Developmental, Promoter Regions, Genetic
- Abstract
Background: Gene expression differences between species are driven by both cis and trans effects. Whereas cis effects are caused by genetic variants located on the same DNA molecule as the target gene, trans effects are due to genetic variants that affect diffusible elements. Previous studies have mostly assessed the impact of cis and trans effects at the gene level. However, how cis and trans effects differentially impact regulatory elements such as enhancers and promoters remains poorly understood. Here, we use massively parallel reporter assays to directly measure the transcriptional outputs of thousands of individual regulatory elements in embryonic stem cells and measure cis and trans effects between human and mouse., Results: Our approach reveals that cis effects are widespread across transcribed regulatory elements, and the strongest cis effects are associated with the disruption of motifs recognized by strong transcriptional activators. Conversely, we find that trans effects are rare but stronger in enhancers than promoters and are associated with a subset of transcription factors that are differentially expressed between human and mouse. While we find that cis-trans compensation is common within promoters, we do not see evidence of widespread cis-trans compensation at enhancers. Cis-trans compensation is inversely correlated with enhancer redundancy, suggesting that such compensation may often occur across multiple enhancers., Conclusions: Our results highlight differences in the mode of evolution between promoters and enhancers in complex mammalian genomes and indicate that studying the evolution of individual regulatory elements is pivotal to understand the tempo and mode of gene expression evolution.
- Published
- 2020
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37. Long Noncoding RNAs: Molecular Modalities to Organismal Functions.
- Author
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Rinn JL and Chang HY
- Subjects
- Animals, Cell Nucleus genetics, Cell Nucleus metabolism, Eukaryotic Cells cytology, Eukaryotic Cells metabolism, Humans, Nucleic Acid Conformation, Promoter Regions, Genetic, RNA metabolism, RNA, Long Noncoding chemistry, RNA, Long Noncoding metabolism, RNA, Messenger chemistry, RNA, Messenger metabolism, Structure-Activity Relationship, Telomerase metabolism, Telomere Homeostasis, Transcription, Genetic, Genome, Protein Biosynthesis, RNA genetics, RNA, Long Noncoding genetics, RNA, Messenger genetics, Telomerase genetics
- Abstract
We have known for decades that long noncoding RNAs (lncRNAs) can play essential functions across most forms of life. The maintenance of chromosome length requires an lncRNA (e.g., hTERC) and two lncRNAs in the ribosome that are required for protein synthesis. Thus, lncRNAs can represent powerful RNA machines. More recently, it has become clear that mammalian genomes encode thousands more lncRNAs. Thus, we raise the question: Which, if any, of these lncRNAs could also represent RNA-based machines? Here we synthesize studies that are beginning to address this question by investigating fundamental properties of lncRNA genes, revealing new insights into the RNA structure-function relationship, determining cis - and trans -acting lncRNAs in vivo, and generating new developments in high-throughput screening used to identify functional lncRNAs. Overall, these findings provide a context toward understanding the molecular grammar underlying lncRNA biology.
- Published
- 2020
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38. Resolving mechanisms of immune-mediated disease in primary CD4 T cells.
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Bourges C, Groff AF, Burren OS, Gerhardinger C, Mattioli K, Hutchinson A, Hu T, Anand T, Epping MW, Wallace C, Smith KG, Rinn JL, and Lee JC
- Subjects
- Autoimmunity, Humans, Polymorphism, Single Nucleotide, CD4-Positive T-Lymphocytes, NF-kappa B
- Abstract
Deriving mechanisms of immune-mediated disease from GWAS data remains a formidable challenge, with attempts to identify causal variants being frequently hampered by strong linkage disequilibrium. To determine whether causal variants could be identified from their functional effects, we adapted a massively parallel reporter assay for use in primary CD4 T cells, the cell type whose regulatory DNA is most enriched for immune-mediated disease SNPs. This enabled the effects of candidate SNPs to be examined in a relevant cellular context and generated testable hypotheses into disease mechanisms. To illustrate the power of this approach, we investigated a locus that has been linked to six immune-mediated diseases but cannot be fine-mapped. By studying the lead expression-modulating SNP, we uncovered an NF-κB-driven regulatory circuit which constrains T-cell activation through the dynamic formation of a super-enhancer that upregulates TNFAIP3 (A20), a key NF-κB inhibitor. In activated T cells, this feedback circuit is disrupted-and super-enhancer formation prevented-by the risk variant at the lead SNP, leading to unrestrained T-cell activation via a molecular mechanism that appears to broadly predispose to human autoimmunity., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2020
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39. Mitoregulin Controls β-Oxidation in Human and Mouse Adipocytes.
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Friesen M, Warren CR, Yu H, Toyohara T, Ding Q, Florido MHC, Sayre C, Pope BD, Goff LA, Rinn JL, and Cowan CA
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Cell Respiration, Conserved Sequence, Energy Metabolism, Humans, Lipid Metabolism, Lipids blood, Mice, Knockout, Mitochondria metabolism, Mitochondrial Proteins chemistry, Oxidation-Reduction, Adipocytes metabolism, Mitochondrial Proteins metabolism
- Abstract
We previously discovered in mouse adipocytes an lncRNA (the homolog of human LINC00116) regulating adipogenesis that contains a highly conserved coding region. Here, we show human protein expression of a peptide within LINC00116, and demonstrate that this peptide modulates triglyceride clearance in human adipocytes by regulating lipolysis and mitochondrial β-oxidation. This gene has previously been identified as mitoregulin (MTLN). We conclude that MTLN has a regulatory role in adipocyte metabolism as demonstrated by systemic lipid phenotypes in knockout mice. We also assert its adipocyte-autonomous phenotypes in both isolated murine adipocytes as well as human stem cell-derived adipocytes. MTLN directly interacts with the β subunit of the mitochondrial trifunctional protein, an enzyme critical in the β-oxidation of long-chain fatty acids. Our human and murine models contend that MTLN could be an avenue for further therapeutic research, albeit not without caveats, for example, by promoting white adipocyte triglyceride clearance in obese subjects., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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40. The Sox2 transcription factor binds RNA.
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Holmes ZE, Hamilton DJ, Hwang T, Parsonnet NV, Rinn JL, Wuttke DS, and Batey RT
- Subjects
- Animals, Base Sequence, Binding, Competitive, DNA metabolism, Male, Mice, Models, Molecular, Mouse Embryonic Stem Cells metabolism, Protein Binding, Protein Domains, RNA, Long Noncoding genetics, RNA, Long Noncoding metabolism, SOXB1 Transcription Factors chemistry, Sequence Deletion, Static Electricity, RNA metabolism, SOXB1 Transcription Factors metabolism
- Abstract
Certain transcription factors are proposed to form functional interactions with RNA to facilitate proper regulation of gene expression. Sox2, a transcription factor critical for maintenance of pluripotency and neurogenesis, has been found associated with several lncRNAs, although it is unknown whether these interactions are direct or via other proteins. Here we demonstrate that human Sox2 interacts directly with one of these lncRNAs with high affinity through its HMG DNA-binding domain in vitro. These interactions are primarily with double-stranded RNA in a non-sequence specific fashion, mediated by a similar but not identical interaction surface. We further determined that Sox2 directly binds RNA in mouse embryonic stem cells by UV-cross-linked immunoprecipitation of Sox2 and more than a thousand Sox2-RNA interactions in vivo were identified using fRIP-seq. Together, these data reveal that Sox2 employs a high-affinity/low-specificity paradigm for RNA binding in vitro and in vivo.
- Published
- 2020
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41. In vivo Firre and Dxz4 deletion elucidates roles for autosomal gene regulation.
- Author
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Andergassen D, Smith ZD, Lewandowski JP, Gerhardinger C, Meissner A, and Rinn JL
- Subjects
- Animals, Female, Male, Mice, Mice, Inbred Strains, Mice, Knockout, Transcriptome, X Chromosome Inactivation, DNA, Complementary genetics, Gene Deletion, Gene Expression Regulation, RNA, Long Noncoding genetics
- Abstract
Recent evidence has determined that the conserved X chromosome mega-structures controlled by the Firre and Dxz4 loci are not required for X chromosome inactivation (XCI) in cell lines. Here, we examined the in vivo contribution of these loci by generating mice carrying a single or double deletion of Firre and Dxz4 . We found that these mutants are viable, fertile and show no defect in random or imprinted XCI. However, the lack of these elements results in many dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, we identified superloop, megadomain, and Firre locus-dependent gene sets. The largest transcriptional effect was observed in all strains lacking the Firre locus, indicating that this locus is the main driver for these autosomal expression signatures. Collectively, these findings suggest that these X-linked loci are involved in autosomal gene regulation rather than XCI biology., Competing Interests: DA, ZS, JL, CG, AM, JR No competing interests declared, (© 2019, Andergassen et al.)
- Published
- 2019
- Full Text
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42. The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis.
- Author
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Lewandowski JP, Lee JC, Hwang T, Sunwoo H, Goldstein JM, Groff AF, Chang NP, Mallard W, Williams A, Henao-Meija J, Flavell RA, Lee JT, Gerhardinger C, Wagers AJ, and Rinn JL
- Subjects
- Animals, Fertility genetics, Gene Expression Regulation, Developmental, Immunity, Innate drug effects, Lipopolysaccharides pharmacology, Mice, Knockout, Organ Specificity genetics, Phenotype, RNA, Long Noncoding metabolism, Genetic Loci, Hematopoiesis genetics, RNA, Long Noncoding genetics
- Abstract
RNA has been classically known to play central roles in biology, including maintaining telomeres, protein synthesis, and in sex chromosome compensation. While thousands of long noncoding RNAs (lncRNAs) have been identified, attributing RNA-based roles to lncRNA loci requires assessing whether phenotype(s) could be due to DNA regulatory elements, transcription, or the lncRNA. Here, we use the conserved X chromosome lncRNA locus Firre, as a model to discriminate between DNA- and RNA-mediated effects in vivo. We demonstrate that (i) Firre mutant mice have cell-specific hematopoietic phenotypes, and (ii) upon exposure to lipopolysaccharide, mice overexpressing Firre exhibit increased levels of pro-inflammatory cytokines and impaired survival. (iii) Deletion of Firre does not result in changes in local gene expression, but rather in changes on autosomes that can be rescued by expression of transgenic Firre RNA. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis.
- Published
- 2019
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43. Linking long noncoding RNA to drug resistance.
- Author
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Smallegan MJ and Rinn JL
- Subjects
- Drug Resistance, Neoplasm, Drug Tolerance, Antineoplastic Agents, RNA, Long Noncoding
- Abstract
Competing Interests: The authors declare no competing interest.
- Published
- 2019
- Full Text
- View/download PDF
44. Differential contribution of steady-state RNA and active transcription in chromatin organization.
- Author
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Barutcu AR, Blencowe BJ, and Rinn JL
- Subjects
- Dactinomycin pharmacology, Genome, Human, Humans, K562 Cells, Models, Biological, Ribonucleases metabolism, Chromatin genetics, RNA genetics, Transcription, Genetic drug effects
- Abstract
Nuclear RNA and the act of transcription have been implicated in nuclear organization. However, their global contribution to shaping fundamental features of higher-order chromatin organization such as topologically associated domains (TADs) and genomic compartments remains unclear. To investigate these questions, we perform genome-wide chromatin conformation capture (Hi-C) analysis in the presence and absence of RNase before and after crosslinking, or a transcriptional inhibitor. TAD boundaries are largely unaffected by RNase treatment, although a subtle disruption of compartmental interactions is observed. In contrast, transcriptional inhibition leads to weaker TAD boundary scores. Collectively, our findings demonstrate differences in the relative contribution of RNA and transcription to the formation of TAD boundaries detected by the widely used Hi-C methodology., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2019
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45. RNA-seq as a tool for evaluating human embryo competence.
- Author
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Groff AF, Resetkova N, DiDomenico F, Sakkas D, Penzias A, Rinn JL, and Eggan K
- Subjects
- Biopsy, Blastocyst metabolism, Female, Humans, Karyotype, Karyotyping, Pregnancy, Sequence Analysis, RNA, Exome Sequencing, Embryo Implantation genetics, Embryonic Development genetics, Fertilization in Vitro, Genetic Testing, Preimplantation Diagnosis methods
- Abstract
The majority of embryos created through in vitro fertilization (IVF) do not implant. It seems plausible that rates of implantation would improve if we had a better understanding of molecular factors affecting embryo competence. Currently, the process of selecting an embryo for uterine transfer uses an ad hoc combination of morphological criteria, the kinetics of development, and genetic testing for aneuploidy. However, no single criterion can ensure selection of a viable embryo. In contrast, RNA-sequencing (RNA-seq) of embryos could yield high-dimensional data, which may provide additional insight and illuminate the discrepancies among current selection criteria. Recent advances enabling the production of RNA-seq libraries from single cells have facilitated the application of this technique to the study of transcriptional events in early human development. However, these studies have not assessed the quality of their constituent embryos relative to commonly used embryological criteria. Here, we perform proof-of-principle advancement to embryo selection procedures by generating RNA-seq libraries from a trophectoderm biopsy as well as the remaining whole embryo. We combine state-of-the-art embryological methods with low-input RNA-seq to develop the first transcriptome-wide approach for assessing embryo competence. Specifically, we show the capacity of RNA-seq as a promising tool in preimplantation screening by showing that biopsies of an embryo can capture valuable information available in the whole embryo from which they are derived. Furthermore, we show that this technique can be used to generate a RNA-based digital karyotype and to identify candidate competence-associated genes. Together, these data establish the foundation for a future RNA-based diagnostic in IVF., (© 2019 Groff et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2019
- Full Text
- View/download PDF
46. Single-cell imaging reveals unexpected heterogeneity of telomerase reverse transcriptase expression across human cancer cell lines.
- Author
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Rowland TJ, Dumbović G, Hass EP, Rinn JL, and Cech TR
- Subjects
- Alleles, Cell Line, Tumor, Cell Nucleus enzymology, Cell Nucleus genetics, Cytoplasm enzymology, Cytoplasm genetics, HEK293 Cells, Humans, In Situ Hybridization, Fluorescence methods, Promoter Regions, Genetic, RNA Splicing, RNA, Messenger genetics, RNA, Messenger metabolism, Single-Cell Analysis, Telomerase metabolism, Telomere metabolism, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Neoplastic, Neoplasms genetics, Telomerase genetics
- Abstract
Telomerase is pathologically reactivated in most human cancers, where it maintains chromosomal telomeres and allows immortalization. Because telomerase reverse transcriptase (TERT) is usually the limiting component for telomerase activation, numerous studies have measured TERT mRNA levels in populations of cells or in tissues. In comparison, little is known about TERT expression at the single-cell and single-molecule level. To address this, we analyzed TERT expression across 10 human cancer lines using single-molecule RNA fluorescent in situ hybridization (FISH) and made several unexpected findings. First, there was substantial cell-to-cell variation in number of transcription sites and ratio of transcription sites to gene copies. Second, previous classification of lines as having monoallelic or biallelic TERT expression was found to be inadequate for capturing the TERT gene expression patterns. Finally, spliced TERT mRNA had primarily nuclear localization in cancer cells and induced pluripotent stem cells (iPSCs), in stark contrast to the expectation that spliced mRNA should be predominantly cytoplasmic. These data reveal unappreciated heterogeneity, complexity, and unconventionality in TERT expression across human cancer cells., Competing Interests: Conflict of interest statement: T.R.C. is on the board of directors of Merck, Inc., and a consultant for Storm Therapeutics, neither of which provided funding for this study.
- Published
- 2019
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47. Illuminating Genomic Dark Matter with RNA Imaging.
- Author
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Raj A and Rinn JL
- Subjects
- Humans, Pattern Recognition, Automated, Genomics methods, RNA, Long Noncoding genetics, Sequence Analysis, RNA methods
- Abstract
In the postgenomic era, it is clear that the human genome encodes thousands of long noncoding RNAs (lncRNAs). Along the way, RNA imaging (e.g., RNA fluorescence in situ hybridization [RNA-FISH]) has been instrumental in identifying powerful roles for lncRNAs based on their subcellular localization patterns. Here, we explore how RNA imaging technologies have shed new light on how, when, and where lncRNAs may play functional roles. Specifically, we will synthesize the underlying principles of RNA imaging techniques by exploring several landmark lncRNA imaging studies that have illuminated key insights into lncRNA biology., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
48. High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue specificity.
- Author
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Mattioli K, Volders PJ, Gerhardinger C, Lee JC, Maass PG, Melé M, and Rinn JL
- Subjects
- Genome, Human, Humans, Organ Specificity, Polymorphism, Single Nucleotide, Promoter Regions, Genetic, RNA, Long Noncoding genetics
- Abstract
Transcription initiates at both coding and noncoding genomic elements, including mRNA and long noncoding RNA (lncRNA) core promoters and enhancer RNAs (eRNAs). However, each class has a different expression profile with lncRNAs and eRNAs being the most tissue specific. How these complex differences in expression profiles and tissue specificities are encoded in a single DNA sequence remains unresolved. Here, we address this question using computational approaches and massively parallel reporter assays (MPRA) surveying hundreds of promoters and enhancers. We find that both divergent lncRNA and mRNA core promoters have higher capacities to drive transcription than nondivergent lncRNA and mRNA core promoters, respectively. Conversely, intergenic lncRNAs (lincRNAs) and eRNAs have lower capacities to drive transcription and are more tissue specific than divergent genes. This higher tissue specificity is strongly associated with having less complex transcription factor (TF) motif profiles at the core promoter. We experimentally validated these findings by testing both engineered single-nucleotide deletions and human single-nucleotide polymorphisms (SNPs) in MPRA. In both cases, we observe that single nucleotides associated with many motifs are important drivers of promoter activity. Thus, we suggest that high TF motif density serves as a robust mechanism to increase promoter activity at the expense of tissue specificity. Moreover, we find that 22% of common SNPs in core promoter regions have significant regulatory effects. Collectively, our findings show that high TF motif density provides redundancy and increases promoter activity at the expense of tissue specificity, suggesting that specificity of expression may be regulated by simplicity of motif usage., (© 2019 Mattioli et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2019
- Full Text
- View/download PDF
49. Interchromosomal interactions: A genomic love story of kissing chromosomes.
- Author
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Maass PG, Barutcu AR, and Rinn JL
- Subjects
- Animals, CCCTC-Binding Factor genetics, Cell Nucleus genetics, Cell Nucleus metabolism, Cell Nucleus ultrastructure, Chromosomes ultrastructure, DNA chemistry, DNA metabolism, Gene Expression Regulation, Gene Regulatory Networks, Humans, Molecular Imaging, Nucleic Acid Conformation, RNA, Long Noncoding genetics, CCCTC-Binding Factor metabolism, Chromosome Aberrations, Chromosomes metabolism, DNA genetics, Genome, RNA, Long Noncoding metabolism
- Abstract
Nuclei require a precise three- and four-dimensional organization of DNA to establish cell-specific gene-expression programs. Underscoring the importance of DNA topology, alterations to the nuclear architecture can perturb gene expression and result in disease states. More recently, it has become clear that not only intrachromosomal interactions, but also interchromosomal interactions, a less studied feature of chromosomes, are required for proper physiological gene-expression programs. Here, we review recent studies with emerging insights into where and why cross-chromosomal communication is relevant. Specifically, we discuss how long noncoding RNAs (lncRNAs) and three-dimensional gene positioning are involved in genome organization and how low-throughput (live-cell imaging) and high-throughput (Hi-C and SPRITE) techniques contribute to understand the fundamental properties of interchromosomal interactions., (© 2018 Maass et al.)
- Published
- 2019
- Full Text
- View/download PDF
50. Enhancers in the Peril lincRNA locus regulate distant but not local genes.
- Author
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Groff AF, Barutcu AR, Lewandowski JP, and Rinn JL
- Subjects
- Animals, Mice, Knockout, Gene Expression Regulation, RNA, Long Noncoding, Regulatory Elements, Transcriptional
- Abstract
Background: Recently, it has become clear that some promoters function as long-range regulators of gene expression. However, direct and quantitative assessment of enhancer activity at long intergenic noncoding RNA (lincRNA) or mRNA gene bodies has not been performed. To unbiasedly assess the enhancer capacity across lincRNA and mRNA loci, we performed a massively parallel reporter assay (MPRA) on six lincRNA loci and their closest protein-coding neighbors., Results: For both gene classes, we find significantly more MPRA activity in promoter regions than in gene bodies. However, three lincRNA loci, Lincp21, LincEnc1, and Peril, and one mRNA locus, Morc2a, display significant enhancer activity within their gene bodies. We hypothesize that such peaks may mark long-range enhancers, and test this in vivo using RNA sequencing from a knockout mouse model and high-throughput chromosome conformation capture (Hi-C). We find that ablation of a high-activity MPRA peak in the Peril gene body leads to consistent dysregulation of Mccc1 and Exosc9 in the neighboring topologically associated domain (TAD). This occurs irrespective of Peril lincRNA expression, demonstrating this regulation is DNA-dependent. Hi-C confirms long-range contacts with the neighboring TAD, and these interactions are altered upon Peril knockout. Surprisingly, we do not observe consistent regulation of genes within the local TAD. Together, these data suggest a long-range enhancer-like function for the Peril gene body., Conclusions: A multi-faceted approach combining high-throughput enhancer discovery with genetic models can connect enhancers to their gene targets and provides evidence of inter-TAD gene regulation.
- Published
- 2018
- Full Text
- View/download PDF
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